In eukaryotic cells actin filaments assemble and disassemble in a dynamic fashion to generate the forces necessary to drive several cellular events such as cell migration, morphogenesis during development and homeostatic processes such as endocytosis. Great progress has been made in elucidating the molecular mechanisms that drive endocytosis in the budding yeast Saccharomyces cerevisiae, and it is well established that the actin cytoskeleton has a central role in endocytic internalization. The Arp 2/3 activator, yeast WASP (Las 17) is critical for the initiation of actin assembly at sites of endocytosis; however, the molecular mechanism by which yeast WASP is recruited to endocytic sites, and how yeast WASP activity is regulated during endocytosis remains largely unknown. This proposal aims to identify the molecular pathway by which yeast WASP is recruited to endocytic sites and the regulatory mechanism of yeast WASP activity during endocytosis. Our first aim will define the minimal region of WASP required for localization at endocytic patches and identify the molecular signals and binding partners that are important for yeast WASP recruitment. Our second aim will identify proteins that work in concert with the F-BAR protein Bzz1 to trigger yeast WASP activity and actin assembly during endocytosis. The endocytic pathway and the regulatory mechanisms that drive actin assembly are highly conserved in higher eukaryotes. Therefore, principles revealed in our studies should shed light on the regulatory mechanisms of actin assembly during several normal and pathological processes.